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Objective: To explore the accuracy of estimated 24-hour urinary iodine excretion(24hUIEest) in assessing iodine nutritional status. Design: Fasting venous blood, 24-hour and spot urine samples were collected during the day. The urinary iodine concentration (UIC) and urinary creatinine concentration (UCrC) were measured, and the urinary iodine-to-creatinine ratio (UI/Cr), 24hUIEest, and 24-hour urinary iodine excretion(24hUIE) were calculated. At the population level, correlation and consistency between UIC, UI/Cr, 24hUIEest and 24hUIE were assessed using correlation analysis and Bland-Altman plots. At the individual level, ROC curves were used to analyse the accuracy of the above indicators for evaluating insufficient and excessive iodine intake. The reference interval of 24hUIEest was established based on percentile values. Setting: Indicator can accurately evaluate individual iodine nutrition during pregnancy remains controversial. Participants: Pregnant women (n=788). Results: Using 24hUIE as standard, the correlation coefficients of 24hUIEest from different periods of the day ranged from 0.409 to 0.531, and the relative average differences ranged from 4.4% to 10.9%. For diagnosis of insufficient iodine intake, the area under the ROC curve of 24hUIEest was 0.754, sensitivity and specificity were 79.6% and 65.4%, respectively. For diagnosis of excessive iodine intake, the area of 24hUIEest was 0.771, sensitivity and specificity were 66.7% and 82.0%, respectively. The reference interval of 24hUIEest was 58.43-597.65μg. Conclusions: 24hUIEest can better indicate iodine nutritional status at a relatively large sample size in a given population of pregnant women. It can be used for early screening at the individual level to obtain more lead time for pregnant women.